LONG-TERM AND FUNCTIONAL CULTURE OF HEPATIC ORGANOIDS (eHEPO) DERIVED FROM EPCAM+ ENDODERMAL PROGENITOR CELLS DIFFERENTIATED FROM INDUCED PLURIPOTENT STEM CELLS
A three-dimensional (3D) liver organoid obtained through the differentiation of induced pluripotent stem cells (IPSCs) in laboratory culture medium and a production method of the 3D hepatic organoid are provided. The production method includes the following steps: differentiating the IPSCs into a definitive endoderm in a medium containing Activin A, Wnt3a, and R-spo1 factors; adding 5 ng/ml R-spo 1 during IPSC differentiation to increase an amount of EpCAM+endoderm progenitor cells; cell sorting of the EpCAM+endoderm progenitor cells through a fluorescence-activated cell sorting (FACS) method; culturing a sorted EpCAM+endoderm progenitor cells in a 3D Matrigel medium of 37° C., 5% CO2, 95% humidity and 7.2-7.5 pH.
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This application is the national phase entry of International Application No. PCT/TR2021/050761, filed on Aug. 2, 2021, which is based upon and claims priority to Turkish Patent Application No. 2020/16056 (TR), filed on Oct. 8, 2020, the entire contents of which are incorporated herein by reference.
SEQUENCE LISTINGThe instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy is named GBUY160_Sequence_Listing.txt, created on 03/23/2023, and is 1,697 bytes in size.
TECHNICAL FIELDThe invention relates to three-dimensional liver organoids obtained through the differentiation of induced pluripotent stem cells (IPSCs) in laboratory culture medium.
BACKGROUNDOrganoids are three-dimensional small structures which are structurally and functionally organ-like, and they are generated under laboratory conditions from organ-specific adult stem cells or pluripotent stem cells.
Organoids form by mimicking the self-organisation processes seen during embryo development. These main processes are: the specific separation of cells (cell sorting out) and the gravitation of the cell towards its future cell type, depending on its spatial location (lineage commitment).
Patent document No US 2018/0258400 A1 relates to the formation of three-dimensional liver organoids from pluripotent stem cells. Liver organoids are obtained from multipotent endoderm spheroid progenitor cells derived from pluripotent stem cells. Endoderm cells are obtained in 8 days; 3D hepatic organoid lineage stage takes 47 days and functional hepatic organoid culture lasts 68 days. The designated organoid culture medium requires more growth factor, which consequently increases the cost. In addition to the growth factors for the organoid stage, there is also a need for the commercially available HCM (LONZA) medium. Not only is this an expensive medium, but the exact amount of its components is not known since it is a commercial product, and it also does not have an alternative. In order to form an organoid, you need to start with a single cell, form 3D spheroid structures, then convert it back to a single cell again to form an organoid. These procedures require a lot of time, effort and expenditure. Furthermore, the functionality and cellular content of the organoids in a late passage is not known, and it is stated that organoids can be cultured for a maximum of 4.5 months. It is necessary to set off with a heterogeneous population to form the organoid culture.
Patent document No US 2019/0298775 A1 relates to the formation of liver organoids from pluripotent stem cells. In addition to the growth factors for the organoid stage, there is also a need for the commercially available HCM (LONZA) medium. Not only is this an expensive medium, but the exact amount of its components is not known as it is a commercial product. There is no data on the functionality and cellular content of the organoids in a late passage. It is necessary to set off with a heterogeneous population to form the organoid culture. Organoid formation last 28 days and can be cultured for a maximum of 30 days more.
Various growth factors and chemicals are used in iPSC-based organoid formation protocols. As not all cells are in the same state during this process, they differentiate in different ways. Some cells differentiate one way at a later stage, whereas others do not differentiate or do so in a less extent. This situation creates a heterogeneous population. Therefore, the result is setting off with cells of different potential during organoid formation, which consequently creates the possible existence of different cell types or progenitors derived from the endoderm in the obtained organoid structure.
For these reasons, there is a need for organoids which are obtainable more quickly, highly efficient, able to be cultured for a long time, able to carry out liver functions for longer periods, cost-effective and not commercially dependent.
BRIEF SUMMARY OF THE INVENTIONThe invention relates to the creation of three-dimensional cell/tissue culture which is known as organoid technology. It involves generating the functional liver (hepatic) organoid culture from EpCAM+ endodermal progenitor cells obtained through the differentiation of induced pluripotent stem cells under laboratory conditions. This culture has superiority over other hepatic organoids obtained from induced pluripotent stem cells (IPSCs) for the following reasons: a) it forms in just 14 days, b) it can be increased in culture in a proper way for over a year, c) it is able to carry out specific liver functions such as albumin production, glycogen storage, cellular ingestion of low-density lipoprotein (LDL) and cytochrome p450 enzyme activity, even in late passages. For this reason, this technology referred to as eHEPO will be rather convenient in terms of personalised drug screening, preclinical hepatotoxicity analyses and disease modelling.
The start to the invention was given by using a homogenous cell group which would allow the isolation of EpCAM positive cells at the endoderm differentiation stage and enable the formation of liver-specific cells only. This shows the source of the organoids and what kind of a population they are.
The invention demonstrates that the organoid formation percentage using EpCAM+ cells isolated from the endoderm is a minimum of 35%, and that EpCAM− cells do not form organoids. The organoid acquisition duration using the present technique is rather long due to the fact that organoids are obtained from all endoderm cells, and the culture cannot be run for a long period of time.
It is possible to obtain hepatic organoids in IPSCs in 14 days. An extra 10 days of differentiation is required for the formation of functional liver hepatocyte-like cells. In other words, hepatocytes with liver-specific functionality such as albumin secretion, cellular ingestion of LDL, fat storage, glycogen storage and drug detoxification enzyme activity are obtained at the end of the 25th day.
In addition, the eHEPO technology was used in the modelling of citrullinemia, a rare metabolic liver disease. Through culturing, it was possible to mimic the exact phenotype seen in humans (like the liver not being able to eliminate ammonia).
The objective of the invention is to generate liver organoids from pluripotent stem cells. This invention enables the formation of liver organoids with the help of skin biopsies taken from healthy volunteers, as well as those with genetic liver diseases. The benefit of its technique is that the organoids are obtainable in a short time, that they still remain normal in long-term cultures and that they have liver functionality as disease phenotypes. Organoids obtained with this technology are advantageous in that they can be used in hepatotoxicity and drugs screenings.
Through this invention, the endoderm can be obtained in 5 days, the 3D hepatic organoid lineage in 10 days and the functional hepatic organoid in 25 days.
The culture of organoids is possible in an endoderm-derived hepatic organoid (eHEPO) culture up to the AT passage 48 for approximately 16-18 months, without losing their functionalities.
The invention involves sorting of the EpCAM cells and starting with a more pure and potential cell population, thus obtaining a more productive culture.
Through the method of the invention, an attempt was made to form organoids with EpCAM+ and EpCAM− cells. The former enabled 35-40% organoid formation, whereas the latter did not form any organoids.
Within the scope of the invention, the characterisation and functionality of organoids were tested from early passage phases to later stages.
With the invention, it is possible to obtain organoids which are not dependent on commercial products like HCM, and which preserve their cellular content and functionality after long-duration cultures.
During organoid formation, EpCAM, the more pure and effective cell, was isolated and, thus, the organoid was formed. This enables a quicker and more effective way of organoid formation.
In addition, this method was used in the modelling of citrullinemia, which is a urea cycle disorder. In terms of the organoids formed from the IPSCs obtained from dermal fibroblasts taken from two citrullinemia patients, it was observed that ammonia elimination mimicking the disease phenotype was not possible.
The invention creates an easy and robust protocol to model all liver metabolic diseases in vitro.
Through this invention, three-dimensional hepatic (liver) organoids were generated to be used in research towards the gene therapy planning and the discovery of drugs for the treatment of all liver metabolic diseases.
The invention specifically was used to mimic Citrullinemia phenotype in vitro and this was recently the most reliable human model for Citrullinemia as a drug screening platform.
The invention can be utilized as a platform for drug hepatotoxicity assay of the drug candidates.
The invention provides a live hepatic function monitoring system to follow any harmful effect on human liver
The invention comprises the operational steps of the production method of three-dimensional hepatic organoids obtained from induced pluripotent stem cells (IPSCs):
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- The differentiation of IPSCs into a definitive endoderm in a medium containing Activin A, Wnt3a, and R-spo1 factors,
- Adding 5 ng/ml R-spo 1 during IPSC differentiation to increase the amount of EpCAM+ endoderm cells,
- Cell sorting of the EpCAM+endodermal progenitor cells through the fluorescence-activated cell sorting (FACS) method,
- Culturing the sorted EpCAM+endodermal progenitor cells in a 3D Matrigel medium of 37° C., 5% CO2, 95% humidity and 7.2-7.5 pH,
- Once the Matrigel solidifies, cell culture medium of 1.25 mM N-acetylcysteine with 1% N2 and 1% B27 without retinoic acid, 10 nM gastrin and 50 ng/ml EGF, 10% RSPO1 is added, 100 ng/ml FGF 10, 25 ng/ml HGF, 10 mM Nicotinamide, 5 uM A8301, DMEM/F12 with added 10 uM FSK culture medium is added; during the first 3 days of this step, 25 ng/ml Noggin and 30% Wnt CM and 10 uM Y27632 is added to the culture medium,
- For the functional hepatocyte differentiation of the organoids, the medium needs to be changed with 1% N2 and 1% B27 without retinoic acid, 10 nM gastrin and 50 ng/ml EGF, 100 ng/ml FGF 10, 25 ng/ml HGF, 500 nM A8301, 10 uM DAPT, 25 ng/ml BMP7 and 30 uM Dexamethasone added developed cell culture medium DMEM/F12 containing differentiation medium.
To start with, IPSCs were differentiated into definitive endoderms in a medium containing 100 ng/mL Activin A, 50 ng/mL Wnt3a, and 5 ng/mL R-spo1factors for 5 days (
Afterwards, EpCAM+ cells obtained as a result of the two-dimensional differentiation of IPSCs were sorted through fluorescence-activated cell sorting (FACS) in medium culturing organoids from adult liver, and the EpCAM+ or EpCAM− endodermal progenitor cells were cultured in 3D Matrigel medium of 37° C., 5% CO2, 95% humidity and 7.2-7.5 pH. IPSC-derived endoderm cell suspensions were counted with Trypan blue to determine the number of live cells, then the cells were thawed 1×106 cell/1 ml with a sorting buffer (1×PBS, 1 mM EDTA, 25 mM HEPES pH 7.0, 1% FBS, 0.2 μm filtered and kept at +4° C.) and strained through a 100 um mesh. Following being centrifuged, the pellet was thawed with 100 ul buffer for 107 cells, and incubated with anti-CD326 (EpCAM)-FITC with a ratio of 1:11 for 10 minutes at 4° C. After the washes, the pellet was thawed 1×106 cell/1 ml with a sorting buffer and sorted with the flow cytometry method. The sorted cells were centrifuged, the obtained cell pellet was resuspended with Matrigel, and then cultured according to the “human liver organoid culture” protocol. 14 days after culturing, organoids larger than 100 μm were scored. Cells were cultured in Matrigel in 48-well (non-stick) cell culture plates as 3000-10000 cell/well. Once the Matrigel had solidified, EM (expansion medium/multiplication and maintenance medium) culture medium was added. EM culture medium consist of 1% N2 and 1% B27 without retinoic acid and 1.25 mM N-acetylcysteine, 10 nM gastrin and 50 ng/ml EGF, 10% RSPO1 conditioned cell culture medium (home-made), 100 ng/ml FGF 10, 25 ng/ml HGF, 10 mM Nicotinamide, 5 uM A8301 and Advanced DMEM/F12 with added 10 uM FSK. 25 ng/ml Noggin and 30% Wnt CM and 10 uM Y27632 were added to the culture medium during the first 3 days. The medium then continued with a culture medium which did not contain Noggin, Wnt and Y27632. Organoids were removed from Matrigel 10-14 days later, and they were mechanically split into pieces and transferred into a fresh matrix. Passaging was conducted every 7-10 days for at least 12 months at a ratio of 1:4-1:8. EpCAM (+) cells, which were defined for the first time by Hans Clevers and his team for the formation of adult human liver organoids and which grow in expansion medium (EM), started forming organoids from approximately day 3, and organoids with diameters larger than 100 μm were scored at the end of day 11. As a result of assays run with EpCAM (−) cells, it was seen that these cells do not have the ability to form organoids. Liver organoids were kept in cell culture medium containing 25 ng/ml BMP7 for 7-10 days in culture conditions explained above. Then the cultures were passaged and maintained 2-4 days more in the same medium. Then the medium was changed with DM (Differentiation medium), which consists of 1% N2 and 1% B27 without retinoic acid, 10 nM gastrin and 50 ng/ml EGF, 100 ng/ml FGF 10, 25 ng/ml HGF, 500 nM A8301, 10 uM DAPT, 25 ng/ml BMP7 and 30 uM Dexamethasone added developed cell culture medium DMEM/F12. This procedure was done every 2-3 days for a period of 10-14 days.
As a result, it was possible to enable the formation of more EpCAM (+) endoderm cells with this invention by means of modifying the endoderm differentiation medium. In addition to this, sorting of these EpCAM (+) cells provided a purer population, and thus, a very fast and effective organoid formation method. Moreover, liver organoids obtained through this method remained in culture longer than 48 passages and do not lose their liver functions even in later cultures.
In a period as short as 1 week, EpCAM+endodermal cells were able to form three-dimensional empty structures resembling liver organoids derived from adult stem cells, whereas EpCAM− cells were deprived of this ability. When culturing EpCAM+derived organoids, structures with a round-shaped organoid morphology which had an approximate diameter of 100 nm and different sides were observed (
In order to advance the maturation of the obtained hepatic cells, organoids were cultured in differentiation medium (DM) for 10-14 days, and the expression of liver-specific genes and structural organisation were analysed, the latter through immunostaining. At this point, immunostaining was conducted for CK18, ZO-1, E-CAD, CK19, ALB and A1AT in DM conditions in both early passage (p10) and late passage (p48) organoids. All differentiated organoids consist of ALB+ and CK18+ hepatocytes which similarly have a typical polar structure, and the ZO-1 expression shows the presence of tight junctions separating apical and basolateral domains. Moreover, the E-CAD staining pattern shows the liver epithelium. ALB and A1AT staining provides evidence for hepatocyte maturation expressed even at late passages. IN the meantime, the presence of CK19+ cells particularly around lumen-like structures demonstrates the presence of cholangiocyte-like and/or progenitor cell population in differentiated organoids (
An albumin-GFP reporter system was developed to be able to characterise more the maturation of eHEPOs from IPSCs. An albumin enhancer/promoter conducting GFP expression was cloned to a lentiviral backbone between two flanking insulator elements, and following the integration into IPSCs, it was possible to observe the real-time differentiation of IPSCs into mature hepatocytes in organoids. Organoids were formed from reporter-carrying IPSCs, and it was identified that they become GFP positive after 5 days in DM culture conditions (
Following the protocol explained above, CTLN organoids that can last longer than 6 months in culture were successfully formed (
Claims
1. A production method of a three-dimensional (3D) hepatic organoid obtained from induced pluripotent stem cells (IPSCs) comprising the steps of:
- differentiating the IPSCs into a definitive endoderm in a medium containing Activin A, Wnt3a, and R-spo1 factors,
- adding 5 ng/ml R-spo 1 during IPSC differentiation to increase an amount of EpCAM+endoderm progenitor cells,
- cell sorting of the EpCAM+endoderm progenitor cells through a fluorescence-activated cell sorting (FACS) method,
- culturing a sorted EpCAM+endoderm progenitor cells in a 3D Matrigel medium of 37° C., 5% CO2, 95% humidity and 7.2-7.5 pH,
- once the 3D Matrigel medium solidifies, a cell culture medium of 1.25 mM N-acetylcysteine with 1% N2 and 1% B27 without retinoic acid, 10 nM gastrin and 50 ng/ml EGF, 10% RSPO1 is added to the 3D Matrigel medium, 100 ng/ml FGF 10, 25 ng/ml HGF, 10 mM Nicotinamide, 5 uM A8301, DMEM/F12 with added 10 uM FSK culture medium is added to the 3D Matrigel medium; during the first 3 days of this step, 25 ng/ml Noggin and 30% Wnt CM and 10 uM Y27632 is added to the 3D Matrigel medium,
- for a functional hepatocyte differentiation of organoids, the 3D Matrigel medium needs to be changed with 1% N2 and 1% B27 without retinoic acid, 10 nM gastrin and 50 ng/ml EGF, 100 ng/ml FGF 10, 25 ng/ml HGF, 500 nM A8301, 10 uM DAPT, 25 ng/ml BMP7 and 30 uM Dexamethasone added a developed cell culture medium DMEM/F12 containing a differentiation medium.
2. A 3D hepatic organoid obtained according to claim 1.
3. The method according to claim 1, wherein the 3D hepatic organoid is obtained through the IPSC differentiation in laboratory culture medium.
4. The 3D hepatic organoid according to claim 2, configured for use in research towards a gene therapy planning and a discovery of drugs for a treatment of all liver metabolic diseases.
5. The 3D hepatic organoid according to claim 2, configured for use in a mimic Citrullinemia phenotype in vitro.
6. The 3D hepatic organoid according to claim 2, configured for use as a platform for a drug hepatotoxicity assay of drug candidates.
7. The 3D hepatic organoid according to claim 2, configured for use as a live hepatic function monitoring system to follow any harmful effect on human liver.
Type: Application
Filed: Aug 2, 2021
Publication Date: Dec 7, 2023
Applicants: DOKUZ EYLUL UNIVERSITESI REKTORLUGU (Izmir), IZMIR BIYOTIP VE GENOM MERKEZI (Izmir), KOC UNIVERSITESI (Istanbul)
Inventors: Serife Esra ERDAL BAGRIYANIK (Izmir), Soheil AKBARI (Izmir), Tevfik Tamer ONDER (Istanbul), Nur ARSLAN (Izmir)
Application Number: 18/247,651